Ch. 15 Transcription: DNA Dependent Synthesis of RNA

Question 1

What is a basic description of transcription?

Answer 1

“RNA synthesis using DNA”

Question 2

What is the process of transcription broken down into (3) and what is it similar to?

Answer 2

Chemistry and process are similar to DNA replication.
1. Initiation
2. Elongation
3. Termination

Question 3

What is transcription performed by?

Answer 3

RNA polymerases perform transcription.

Question 4

Where does transcription begin versus replication?

Answer 4

Transcription starts at a promoter. DNA Replication starts at the ori.

Question 5

What are RNA polymerases?

Answer 5

They are enzymes that catalyze that formation of phosphodiester bonds between ribonucleotides.

Question 6

What is the bacterial RNA pol made of?

Answer 6

The bacterial RNA pol is a holoenzyme made of five polypeptide subunits (ɑɑββʹω).

Question 7

What does the bacterial RNA pol holoenzyme associate with and what does it do?

Answer 7

Holoenzyme associates with a 𝜎 subunit, which recognizes a promoter. (Different types of 𝜎 recognize different types of promoters)

Question 8

What are the three kinds of eukaryotic RNA pol? Define each.

Answer 8

RNA pol I: transcribes large rRNA precursors
RNA pol II: transcribes most protein coding RNA (mRNA)
RNA pol III: transcribes small functional RNAs (tRNA, small nuclear RNA, 5S rRNA)

Question 9

What does the similarities between bacterial and eukaryotic RNA polymerases suggest?

Answer 9

Bacterial and eukaryotic pol’s are structurally and functionally similar, suggesting an ancient origin.

Question 10

What direction does RNA pol synthesize in?

Answer 10

Synthesize from 5’→3’ end (only add to a 3’ end).

Question 11

What does RNA pol require to transcribe; what doesn’t it need?

Answer 11

RNA pol doesn’t require a primer, but it does require a template.

Question 12

How many DNA strands are templates for RNA pol?

Answer 12

In transcription, only ONE DNA strand of a gene serves as a template.

Question 13

What is the template strand in transcription?

Answer 13

The DNA strand that is copied. It will be complementary to the RNA transcript.

Question 14

What is the coding strand in transcription?

Answer 14

The DNA strand NOT copied. It will have the same sequence as the RNA transcript, except U will be T.

Question 15

What is the general transcription process (not specifically bacterial or eukaryotic)? (6)

Answer 15

1. RNA pol binds a gene’s promoter (transcription factors are often involved)
2. RNA pol creates a transcription bubble (open complex) in the DNA to gain access to bases
3. The first RNA nucleotide is added at the +1 site
4. Elongation occurs through base pairing and phosphodiester bond formation
5. DNA reanneals (becomes ds again) and RNA is single stranded
6. transcription ends at a terminator site

Question 16

What three things to bacterial promoters contain?

Answer 16

Contains ﹣35 and ﹣10 regions with A’s and T’s. Contains a +1 transcriptional start site. Contains a UP element which is recognized by an ɑ subunit to stabilize RNA pol binding.

Question 17

What elements of the promoter affect transcription in bacteria? (3)

Answer 17

Transcription is affected by the specific ﹣35 and ﹣10 sequence, the size of the spacer, and the UP element.

Question 18

What happens upon binding of a promoter in bacterial transcription?

Answer 18

Upon binding a promoter, the DNA is initially in a closed complex that will convert to an open complex.

Question 19

Where are the coding and template strands separated in bacterial transcription?

Answer 19

Coding and template strands are separated in the ﹣11 to +3 region of the DNA (~14 bases).

Question 20

How does RNA pol gain access to all of the nucleic acids/components involved?

Answer 20

There are several channels in the structure that provide access to the inside of the pol. (DNA entry, DNA exit, RNA exit, nucleotide entry)

Question 21

What is the process of bacterial transcription? (6)

Answer 21

1. DNA enters the RNA pol and a pin separates strands
2. NTPs bind the template DNA strand
3. the pol catalyzes phosphodiester bond formation
4. once a long enough initial RNA is made, transcription enter the elongation stage
5. RNA pol has some proofreading ability which allows it to remove a mismatched base (not as good as DNA proofreading, so there is a higher error rate)
6. Termination of transcription occurs at certain base sequences (⍴-independent or ⍴-dependent)

Question 22

How does RNA pol catalyze phosphodiester bond formation?

Answer 22

RNA pol can catalyze a bond between two free NTPs binding at the +1 and +2 sites, then add additional NTPs.

Question 23

Is the beginning of RNA pol’s activity strong? What is the phenomenon called?

Answer 23

Not really. There is a high probability that the interaction is NOT strong, and the pol may release a short RNA prior to completion: abortive initiation.

Question 24

Does RNA pol stop after an abortive initiation?

Answer 24

No. The pol can and does restart.

Question 25

When is a transcript stable and free from abortive initiation?

Answer 25

Once it reaches 10 bp.

Question 26

Why are the higher error rates of RNA pol tolerated by cells? (2)

Answer 26

Higher error rate is tolerated because many transcripts are produced and RNA is rapidly degraded.

Question 27

How many bases does bacterial RNA pol add per second?

Answer 27

50﹣90 bases per second.

Question 28

What is ⍴-independent termination?

Answer 28

Bacterial transcription termination method. A terminator hairpin loop forms in DNA and disrupts the RNA pol structure to release RNA.

Question 29

What is ⍴-dependent termination?

Answer 29

Bacterial transcription termination method. Transcription of the rut site attracts a ⍴ helicase that moves 5'→3' on the RNA, and disrupts the pol to release RNA.

Question 30

Are all eukaryotic genes transcribed by the same RNA pol?

Answer 30

No. Each type of eukaryotic RNA pol transcribes a certain type of gene.

Question 31

What do all three eukaryotic RNA pol's use for initiation?

Answer 31

All three use TATA-binding protein (TBP) to bind a ﹣30 TATAAA region.

Question 32

What if the eukaryotic gene doesn't have a TATA region, how will TBP bind?

Answer 32

Not all genes have a TATA region. Those that don't will recruit TBP-associated factors that recognize other sequences and attract TBP.

Question 33

How are eukaryotic RNA pol's unique from each other? (2)

Answer 33

Each pol has unique promoters and transcription factors.

Question 34

What is needed for eukaryotic transcription to be initiated? (2)

Answer 34

Multiple transcription factors are required. Also, chromatin structure often needs to be changed to allow RNA pol access to bind.

Question 35

What is the process of eukaryotic transcription initiation? (5)

Answer 35

1. RNA pol and transcription factors (TF) bind an area called the core promoter 2. Multiple TFII proteins bind to their specific sites near a promoter, creating a closed complex 3. TFII has helicase activity and creates the open complex 4. Abortive transcripts may be produced before entering the elongation phase 5. RNA pol II needs to be phosphorylated (>30 proteins required to carry out initiation!)

Question 36

Why is the binding of transcription factors during eukaryotic transcription initiation more complex than bacterial?

Answer 36

Additional sites for transcription factor binding may be thousands of bases away from the core promoter.

Question 37

Why must RNA pol II be phosphorylated after initiation is complete?

Answer 37

RNA pol II must be phosphorylated before it can disengage from the promoter and enter the elongation stage.

Question 38

What is the mediator complex and why is it of note?

Answer 38

A multiprotein mediator complex is required for interaction between transcription factors and regulatory proteins that control transcriptional activity in eukaryotes.

Question 39

When does elongation of eukaryotic transcription begin?

Answer 39

The cell enters elongation phase once RNA pol II creates a non-abortive transcript and has been phosphorylated.

Question 40

Describe eukaryotic transcription elongation.

Answer 40

Elongation factors bind and RNA pol II catalyzes phosphodiester bond formation between NTPs.

Question 41

Why are elongation factors binding during eukaryotic transcription elongation and how do they accomplish this (3)?

Answer 41

Elongation factors bind to and enhance RNA pol II activity by... 1. Suppressing pauses 2. Enhancing editing of mismatches 3. Recruiting proteins involved in post-transcriptional processing of RNA

Question 42

How do RNA pol I and RNA pol III terminate transcription?

Answer 42

They terminate at specific DNA sequences.

Question 43

How does RNA pol II terminate transcription? (2)

Answer 43

RNA pol II doesn't have a specific site. 1. one model is similar to ⍴-independent in bacteria, where hairpin formation destabilizes pol II 2. torpedo model: suggests proteins cut the transcript and disrupt RNA pol II similar to ⍴-dependent in bacteria

Question 44

Regardless of the termination mechanism in eukaryotes, where will termination occur after?

Answer 44

Either mechanism will occur AFTER and AAUAAA site, which is where poly(A) tails will be added.